The present invention relates to a graft animal model for propagating HPV and for evaluating and testing candidate therapeutic agents against HPV. The animal model comprises, a recipient animal engrafted with injured skin graft infected with a host-specific papilloma virus (PV). The grafted skin, having demonstrable papillomas supports the propagation of its host-specific PV. The invention particularly relates to a highly reproducible xenograft animal model for hosting and propagating human papillomavirus, thereby providing a means for generating infectious human PV suspensions and for passaging papillomavirus. The invention additionally relates to a novel method for generating the xenograft human animal model.
Papillomaviruses (PV) are non-enveloped DNA viruses that induce hyperproliferative lesions of the epithelia. The papillomaviruses are widespread in nature and have been recognized in higher vertebrates. Viruses have been characterized, amongst others, from humans, cattle, rabbits, horses, and dogs. The first papillomavirus was described in 1933 as cottontail rabbit papillomavirus (CRPV). Since then, the cottontail rabbit as well as bovine papillomavirus type 1 (BPV-1) have served as experimental prototypes for studies on papillomaviruses. Most animal papillomaviruses are associated with purely epithelial proliferative lesions, and most lesions in animals are cutaneous. In the human there are more than 75 types papillomavirus (HPV) that have been identified and they have been catalogued by site of infection: cutaneous epithelium and mucosal epithelium (oral and genital mucosa). The cutaneous-related diseases include flat warts, plantar warts, etc. The mucosal-related diseases include laryngeal papillomas and anogenital diseases comprising cervical carcinomas (Fields, 1996, Virology, 3rd ed. Lippincottxe2x80x94Raven Pub., Philadelphia, N.Y.).
There are more than 25 HPV types that are implicated in anogenital diseases, these are grouped into xe2x80x9clow riskxe2x80x9d and xe2x80x9chigh riskxe2x80x9d types. The low risk types include HPV type 6, type 11 and type 13 and induce mostly benign lesions such as condyloma acuminata (genital warts) and low grade squamous intraepithelial lesions (SIL). In the United States there are 5 million people with genital warts of which 90% is attributed to HPV-6 and HPV-11. About 90% of SIL are also caused by low risk types 6 and 11. The other 10% of SIL are caused by high risk HPVs.
The high risk types papillomaviruses are associated with high grade SIL and cervical cancer and include most frequently HPV types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from low-grade SIL to high-grade SIL is much more frequent for lesions that contain high risk HPV-16 and -18 as compared to those that contain low risk HPV types. In addition, only four HPV types are detected frequently in cervical cancer (types 16, 18, 31 and 45). About 500,000 new cases of invasive cancer of the cervix are diagnosed annually worldwide (Fields, 1996, supra).
Treatments for genital warts include physical removal such as cryotherapy, CO2 laser, electrosurgery, or surgical excision. Cytotoxic agents may also be used such as trichloroacetic acid (TCA), podophyllin or podofilox. Immunotherapy is also available such as Interferon or Imiquimod. These treatments are not completely effective in eliminating all viral particles and there is either a high cost incurred or uncomfortable side effects related thereto. In fact, there are currently no effective antiviral treatments for HPV infection, since with all current therapies recurrent warts are common (Beutner and Ferenczy, 1997, Amer. J. Med., 102(5A): 28-37).
The life cycle of HPV is closely coupled to keratinocyte differentiation. Infection is believed to occur at a site of tissue disruption in the basal epithelium. Unlike normal cells, cellular division continues as the cell undergoes vertical differentiation. As the infected cells undergo progressive differentiation the viral copy number and viral gene expression increase, with the eventual late gene expression and virion assembly in terminally differentiated keratinocytes and the release of viral particles (Fields, 1996, supra).
Papillomaviruses are fastidious viruses that cannot be propagated in vitro. As such, the virus requires a host-specific animal for growth. The ineffectiveness of the current methods to treat PV infections has demonstrated the need to identify new therapeutic agents as a means to prevent and treat HPV infections. The success of developing candidate therapeutic agents to combat papillomavirus has been limited in part due to difficulties including, propagating the virus, obtaining sufficient infectious viral particles and the lack of a good in-vivo model to evaluate the effectiveness of candidate therapeutic agents. Attempts to overcome these difficulties have been addressed by generating xenograft animal models for human papillomavirus. However, all the models known in the prior art have had limited success in overcoming these difficulties.
The ideal animal model is described as having the following attributes: being widely available, easy to handle and maintain in a laboratory, large enough to provide tissue samples, able to induce and form papilloma lesions that are comparable to those in humans, the papillomas should be readily accessible for treatment, and able to yield a large amount of infectious viral particles (Stanley, et al., 1997, Antiviral Chemistry and Chemotherapy, 8(5):381-400).
In U.S. Pat. Nos. 4,814,268 and 5,071,757 (Kreider et al.), human skin tissue subjected to human papillomavirus was grafted under the renal capsule of athymic mice. This is a complex procedure which requires surgical refinement. The graft is allowed to remain in the animal until recoverable quantities of the virus are produced. Examination of the graft site and recovery of viral particles requires the animals to be killed. The infectivity of the recovered viral particles from the graft site was reported to be only at a 10xe2x88x922 dilution. More importantly, since the papillomas formed are not visible, evaluation of therapeutic agents necessitates sacrificing the animal. Subsequent attempts by this group (Kowett et al., 1990, Int. Virology, 31:109-115) to replicate these published results, harvesting infectious viral stock capable of infecting other animal models, have failed. The authors hypothesized that the first wart tissue collected from patients and used to infect an animal model probably contains more infectious virions and is thus successful in initiating papilloma infection in the xenograft animal.
Bonnez W. et al. (1993, Virology 197:455-458) described human foreskin infected in vitro with HPV type 11, implanted under the renal capsule, peritoneum and subcutaneous in SCID mice. Only 58% of the grafts showed signs of HPV infections. In the subcutaneous implanted grafts, only 25% were positive for HPV by immunocytochemistry and RT-PCR. The resultant subcutaneous papillomas were not serially passaged or harvested.
Brandsma J. L. et al. (1995, J. of Vir. 69:2716-2721) and U.S. Pat. No. 5,811,632, describe the delivery of HPV type 16 genomic DNA to human foreskin engrafted onto SCID mice. In total 16 grafts were inoculated with naked HPV DNA, eight inoculated pre-engrafting and eight post-engrafting. Only two grafts inoculated post-grafting appeared to develop signs of HPV infection. However these two prior art documents do not teach harvesting infectious viral particles or the passaging of papillomavirus.
Sexton C. J. et al. (1995, J. of Gen. Vir. 76:3107-3112) described a grafting method whereby a glass cover slip was first inserted into the graft site of a SCID mouse for one to two weeks. This is replaced with a silicone grafting chamber in which benign wart tissue was placed. After five weeks, macroscopic warts developed. Attempts to graft the wart tissue resulted in hyperproliferative human epithelium devoid of viral infection. Thus serial passaging of these warts and harvesting infectious particles are not taught.
Bonnez W. et al. (1998, J. Virol. 72:5256-5261) reported the isolation and propagation of HPV-16. The virus was isolated from clinical samples and used to infect human foreskin prior to subcutaneous implantation into SCID mice. The sites were prepared by inserting glass cover slips at the graft sites two weeks prior to engrafting the infected foreskin. The lesions at the graft sites were exposed four weeks after engrafting and the animals sacrificed 24 weeks after engrafting. Only three of the five grafts showed small papillomas. The virions from these papillomas were harvested and used to inoculate a second set of xenografted human tissue. In this second set of animals 60 grafts were attempted, the resultant lesions were not exposed and the animals were sacrificed 16 weeks after engrafting. Of the 60 grafts, 34 were positive for the presence of HPV DNA and only 1 was positive for HPV capsid by immunochemistry. This prior art does not teach passaging of papillomas or the potential to harvest virulent infectious viral particles to generate an infectious viral suspension. In this model it took 40 weeks to produce one graft site in which potentially infectious viral particles could be detected. In an improved animal model it would be desirable to markedly decrease the incubation time for inducing papillomas having infectious viral particles and more importantly to increase the success rate of papilloma formation evaluated by an increase in size and number of papillomas.
To date there are no animal models for human papillomavirus infections that are easy to generate, dependable, reliable and reproducible and which allow for serial passaging of papillomas and harvesting of infectious viral particles. There thus remains a need to develop an animal model in which a human papillomavirus can be easily propagated and serially passage without requiring complex surgical procedure, and which produces a great number of papillomas and infectious viral particles suspension.
The animal model of this invention is particularly useful for supporting the complete cycle of viral infection and vegetative growth, and, for selecting and testing candidate agents for the treatment or prevention of papillomavirus infections that would have physiological and pharmacological relevance in humans.
The model of the present invention produces highly reliable and reproducible papillomas from which infectious viral particles can be harvested. The animal model of this invention can further be used for screening and selecting candidate agents for the treatment or prevention of human papillomavirus infections and any conditions caused thereof.
It is a critical feature of the present invention, to provide a method for producing a xenograft animal model wherein injuring the host skin prior to grafting advantageously provides wound healing that fosters papilloma induction. It is a specific advantage of this invention to provide this injury by way of meshing, additionally providing stretching of the host skin to cover a larger graft area, thus reducing the demand for host skin tissue. Further, meshed engrafted tissue improves the survival and health of the engrafted skin tissue.
Therefore, it is a feature of the present invention, to provide a xenograft animal model, which may be used for the growth and propagation of papillomavirus. Particularly, these xenografted animals when infected with a papillomavirus form papillomas as an indication of papillomavirus infection. These animals are a superior model for induction of papillomavirus infection that is reliable and reproducible when compared with other known xenograft animal models.
It is a specific feature of the present invention, to provide human xenografted animals which may be used for the induction, growth and propagation of human papillomavirus, and from which infectious viral particles can be harvested thereby providing infectious viral stock suspension.
It is a further feature of the present invention, to provide such a viral stock suspension to be serially passaged to papillomavirus-free animals in order to induce papillomavirus infections in subsequent xenografted animals.
It is still another feature of the present invention to provide a method for the production of these xenografted animals in order to induce papillomavirus infections in these xenografted animals and in which papillomavirus can be harvested and propagated, and can be passaged to papilloma-free xenografted animals.
A further feature of the present invention is to provide a xenograft animal model to test potential therapeutic agents against papillomavirus infection.
The present description refers to a number of documents, the content of which is incorporated herein by reference.
Thus, the present invention is directed to a graft animal model for reproducible papilloma induction, and propagation of papillomavirus. This model serves also for screening and selecting a therapeutic agent against papillomavirus infection. The invention further provides a method for producing the grafted animal and the model thereby produced.
Therefore and in accordance with a first embodiment of the present invention there is provided a graft animal model for the induction and formation of papillomas, and for the propagation of human papillomavirus which is characterized by:
a recipient animal grafted with host skin tissue, said skin tissue having been injured prior to said grafting,
inoculating said grafted skin tissue with an inoculum of a host-specific papillomavirus,
wherein said grafted skin is supported by said recipient animal and is capable of inducing and sustaining growth of host-specific papillomavirus and harboring at least one papilloma containing infectious viral particles.
The success of the model of the present invention is based on the realization by the Applicant that the process of tissue healing following injury in the donor skin improves the tissue""s susceptibility to PV infection and favors wart formation.
Within the model according to this first embodiment, there is comprised a recipient animal grafted with host skin tissue, wherein said skin tissue has been injured prior to said grafting, whereby said grafted skin is capable of inducing and sustaining growth of host-specific papillomavirus and harboring at least one papilloma containing infectious viral particles.
In accordance with a second embodiment of the present invention, there is provided a method for producing a graft animal model for propagating infectious papilloma viral particles, said method comprising the following steps:
obtaining skin tissue from a host donor and injuring said skin,
grafting said injured skin tissue onto a recipient animal capable of accepting said skin tissue,
inoculating said grafted tissue with an inoculum of a host-specific papillomavirus, and
providing sufficient time for said papillomavirus to propagate in said grafted tissue and to form papillomas as an indication of papillomavirus infection.
An important aspect of this second embodiment is provided in the step of inducing tissue healing following injury in the host skin tissue to be grafted.
In a particular aspect of this second embodiment, inoculation of the injured donor skin tissue with a papillomavirus inoculum can be accomplished using for example papillomavirus suspension that can be applied either in-vitro or in-situ. Injured donor skin tissue inoculated in-vitro, pre-grafting can be engrafted cutaneously or subcutaneously onto the immuno-deficient recipient animal. Injured donor skin tissue that is engrafted cutaneously can also be inoculated in-situ post-grafting.
In a further aspect of the present embodiment, the subcutaneous papillomas formed in the infected grafted animal, can be exposed by cutting open the subcutaneous papillomas with an incision to the skin at the site of the subcutaneous papilloma growth. The exposed papilloma develops a morphology that is similar to cutaneous papilloma and can be observed and evaluated without having to anesthetize or kill the grafted animal.
In accordance with a third embodiment of the present invention, there is provided an graft animal model for screening candidate therapeutic agents for protecting, preventing or treating papillomavirus infection. Accordingly, a candidate agent (in a therapeutically effective amount and in admixture with a pharmaceutical carrier) is administered to the graft animal model of the present invention. The efficacy of the candidate agent is evaluated by means comprising; a change in size, growth and morphology of the papillomas, and/or a decrease in viral load and infectivity, when compared to a control papilloma from an untreated grafted animal.
Therefore, in accordance with a fourth embodiment of the present invention there is provided a method for evaluating the efficacy of a therapeutic agent useful against papilloma virus infection comprising the steps of:
providing a grafted animal model according to the present invention,
inoculating said grafted host skin tissue with an inoculum of host-specific papilloma virus,
treating said papillomavirus-infected animal by administering a candidate therapeutic agent in an appropriate pharmaceutical carrier, and
evaluating the efficacy of said therapeutic agent in preventing the appearance, reducing the physiological symptoms or reducing the evidence of said infection in said infected animal.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of the preferred embodiments with reference to the accompanying drawings which is exemplary and should not be interpreted as limiting the scope of the present invention.